RU2519713C2 - Chelate amphiphilic polymers - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to a chelate amphiphilic polymer as a carrier, a particle as a carrier containing a self-assembling structure of a chelating amphiphilic polymer (polymersome), contrast agents for CEST MRT, SPECT, PET or Spectral CT, containing said particle, and a method of producing the particle. The chelate amphiphilic polymer is capable of aggregation and contains a hydrophilic block (M_A), having a chelating moiety (X) as a terminal group and a hydrophobic block (M_B), wherein the polymer has the formula X-[M_A]_n - [M_B]_m, (i), where n and m are integers ranging from 3 to 1000000, which represent the number of monomer links forming the corresponding blocks. The hydrophilic block is selected from polyethylene oxide, polymethacrylic acid, polyacrylamide derivatives, polyvinyl alcohol or polyhydroxyethylmethacrylate, hydrophilic polypeptides and sugar derivatives. The hydrophobic block is selected from polybutadiene, polyisoprene and polyethylethylene. The chelating moiety is selected from a group comprising polyphosphates, amino carboxylic acids, 1,3-diketones, hydroxy carboxylic acids, polyamines, amino alcohols, aromatic heterocyclic bases, phenols, amino phenols, oximes, peptides containing proximal chelate functional groups, Schiff bases, tetrapyrroles, sulphur compounds, synthetic macrocyclic compounds, phosphonic acid or a combination of two or more of said compounds.

EFFECT: invention provides chelate amphiphilic polymers which are capable of self-assembling and are suitable for use in vulcanisation methods.

14 cl, 7 dwg, 4 ex

Description

FIELD OF THE INVENTION

The invention relates to amphiphilic polymers and nanocarriers, such as the polymers obtained from them, suitable for use as visualizing contrast agents. In particular, the invention relates to contrast agents T ₁ and / or T ₂ for magnetic resonance imaging (MRI), to radioactively labeled compounds for single photon emission computed tomography (SPECT) or positron emission tomography (PET), to elements with large atomic number for (spectral) computed tomography (spectral CT) and contrast media for MRI with saturation transfer depending on chemical exchange (CEST). More specifically, the invention relates to drug delivery under visual control based on polymersomes as drug carriers.

BACKGROUND OF THE INVENTION

A reference to the source regarding amphiphilic compounds, which may include, for example, a radioactive isotope or an MP active metal, is, for example, V. Torchilin, Chemtech 1999, Volume 29, Number 11, 27-34. This publication refers to polychelate amphiphilic polymers. These polymers mainly include poly-L-lysine-based polymers containing a hydrophilic residue with several chelate groups and relatively short, highly lipophilic phospholipid residues. The latter serve to incorporate the polymer into liposomes and micelles.

The invention relates to various classes of amphiphilic polymers, namely, those that are capable of independently aggregating into polymersomes, micelles, or emulsions stabilized by polymers. These polymers can generally be described as polymer blocks containing at least one hydrophilic block (A), preferably having a chain with a molecular weight of more than 500 g / mol, and at least one hydrophobic block (B) , also in the form of a polymer block (i.e., not a lipid). These polymers can take the form of a block copolymer AB, a tri-block polymer ABA or BAB, or any additional polymer block having a terminal hydrophilic block and an end hydrophobic block, including polymers containing chain (C) having fuzzy solvent properties (i.e., neither hydrophilic nor hydrophobic), for example, an ACB ternary copolymer block. Basically, this will mean that block C forms either a new hydrophilic block together with block A, or a new hydrophobic block together with block B.

As a result of the presence of hydrophilic and hydrophobic blocks, amphiphilic polymers have the ability to form self-aggregated structures. The most typical self-aggregated structures are micelles and polymersomes formed in an aqueous environment. In any case, however, depending on the environment in which they are formed, any type of block (i.e. hydrophilic or hydrophobic) can form an internal or external space. With respect to micelles, the inner space implies concentration at one point, the direction of the polymer chains inward, and the outer space contains diverging, outwardly directed polymer chains. In relation to polymers, self-aggregated structures contain a shell defining a cavity. The shell, most resembling liposomes, is formed by a polymer bilayer in an aqueous medium with hydrophobic blocks directed to each other in the inner part of the bilayer, and hydrophilic blocks in the inner part of the cavity and on the outer surface by polymers.

Compared to lipid carriers (i.e. liposomes), polymersomes are chemically more stable, less fluid, less susceptible to interference with biological membranes and less dynamic due to their lower critical aggregation concentration. The result of these properties is a lower degree of opsonization and a longer circulation time. Liposomes, on the other hand, provide the advantage of easily incorporating radioactive or target compounds into the lipid layer. Liposomes can also be successfully used as contrast agents, in which case they are equipped, for example, with a paramagnetic label for MRI or a radioactive isotope for SPECT or PET.

Although liposomes are a very universal approach, the main limitation is the low degree of PEGylation, i.e. the ability to provide on the surface of the covalently bound poly (ethylene glycol). PEGylation is a known method of masking particles introduced into the body of an individual, such as therapeutic proteins, from an individual's immune system. It is believed that this is based on a lower degree of opsonization, as a result of which PEGylated surfaces are less susceptible to macrophage uptake. This provides an increase in the circulation time of the pegylated molecule. Thus, for liposomes and other nanocarriers, which are essentially suitable, masking in a similar manner is desirable, i.e. provision, pegylated nanocarriers.

SUMMARY OF THE INVENTION

It is preferable to provide amphiphilic polymers having the ability to form self-aggregating structures containing a hydrophilic block and a hydrophobic block, where the hydrophilic block provides for the presence of a chelating fragment as an end group. In particular, it is preferable to provide masking (covering) of the structures from the immune system, which can be accomplished using labels for use in imaging methods.

Preferably, it is desirable to provide such materials, and even better to ensure the inclusion of labels, such as metal ions or elements with a large atomic number, paramagnets or radioactive labels.

In order to more effectively ensure the above preferences, the invention relates to a chelated amphiphilic polymer that is capable of self-aggregation (into a polymersome, micelle or into a polymer-stabilized emulsion).

In one aspect, the amphiphilic polymer is implemented as a polymer containing a hydrophilic block, in particular a poly (ethylene oxide) block, and a hydrophobic block, where the hydrophilic block includes a chelating fragment as an end group.

In another aspect, a polymer particle (also referred to as a nanocarrier) is provided as a particle with a self-aggregating structure, such as a polymer-stabilized emulsion (i.e., an oil-in-water emulsion, where the polymer forms a layer around oil droplets) , a micelle or a bilayer defining a cavity (polymersome), where the polymer is an amphiphilic polymer containing a hydrophilic block and a hydrophobic block, where the hydrophilic block includes a chelating fragment as an end group.

In another aspect, the contrast agent for MRI is presented in the form of a nanocarrier containing a self-aggregating structure, as described earlier in this document, where the chelating fragment on the outer surface of the nanocarrier is associated with a paramagnetic metal.

In a further aspect, a chemical exchange saturation transfer contrast agent (CEST) for magnetic resonance imaging (MRI) is presented as a substance containing polymersomes containing a polymer framework defining a cavity, where the cavity contains a stock of protons for analysis, and where the framework allows proton diffusion for analysis; the framework, being an amphiphilic polymer, contains a hydrophilic block and a hydrophobic block, where the hydrophilic block includes a chelating fragment as end group, and where the paramagnetic metal is associated with a chelating fragment in the internal space of the cavity.

In yet another aspect, the radioactive labeling compound used in single photon emission computed tomography (SPECT) or in positron emission tomography (PET) is provided in the form of a nanocarrier containing self-aggregated structures, as described previously herein, where a chelating fragment is on the outer surface nanocarrier and / or in the internal space of the cavity are associated with a radioactive isotope.

In another aspect, a contrast agent for imaging with a (spectral) CT scan is provided in the form of a nanocarrier containing self-aggregated structures, as described earlier in this document, where a chelating fragment on the outer surface of the nanocarrier and / or in the interior of the cavity is associated with a large atomic substance number (for example, with an element such as heavy metal).

The invention additionally includes methods for the preparation and use of amphiphilic chelated polymers with a specific method of use involving drug delivery.

DETAILED DESCRIPTION OF THE INVENTION

In a broad sense, the invention can be described as a chelating amphiphilic polymer capable of self-aggregation. Different from other nanocarriers, the polymer according to the invention is itself capable of chelating, i.e. the formation of a coordination complex with a metal ion. Different from other chelating polymers, the polymer according to the invention is able to form self-aggregated structures (for example, to form polymersomes, instead of being attached to an existing liposome), which makes it suitable to be used as a nanocarrier.

In the most understandable form, the polymer can be described with reference to three main functional elements: a hydrophobic block, which has the property of being torn off by the aqueous medium, a hydrophilic block, which has the property of establishing contact with the aqueous medium, and a chelating fragment, which is the end group of the hydrophilic block, . on the remaining functional residue of the terminal monomer of the hydrophilic block. One can imagine that the polymer chain contains additional reactive side groups, which can also be provided with chelating fragments, but, in this case, the disadvantage is the impossibility under normal conditions to provide 100% modification of the side groups, and thus, it turns out that the polymer is required will contain reactive, usually charged, side groups. In the case of materials based on polylysine in real conditions, this can adversely affect the ability to self-aggregate.

A chelating moiety can be provided with a metal so as to form a coordination complex and thus provide a substantially 'metallized' polymer. Depending on the metal of interest, labeled polymers can be used as contrast agents for MRI (T ₁ , T ₂ , CEST), radionuclide imaging (SPECT, PET) or spectral CT.

This can be attributed to micelles, where the hydrophobic ends are directed to the center, and the hydrophilic ends are spatially spaced. In the case of micelles, the metal can be introduced to form a coordination complex with a chelating fragment either before or after the formation of the micelle, which is essentially the same as a result.

The invention also relates to polymersomes where an amphiphilic polymer in an aqueous environment is provided in the form of a bilayer defining a cavity. In this document, the hydrophobic blocks are directed relative to each other in the inner region of the bilayer boundary, and the hydrophilic blocks are directed both towards the aqueous medium and towards the inner cavity. In this case, there are two fundamentally different ways of forming coordination complexes containing a metal with an amphiphilic polymer. In the first case, polymersomes are first allowed to form, and then metal is provided. In this case, there is no metal bound to the polymer in the inner cavity of the polymersome. In another method, metal is first provided so that substantially all chelating moieties form a coordination complex and then polymersomes are formed. In this case, the metal involved in the interaction is present both on the inner surface of the polymersome (i.e., the inner shell of the cavity) and on the outer surface of the polymersome. In the latter case, depending on the coordination chemistry used, it is also possible to remove the metal from the outer surface or replace it with another metal to ensure the presence of the metal on the outer surface. This provides the desired design flexibility, for example, allowing polymersomes to perform various functions depending on various methods.

The preparation of a polymersome or micelle in an aqueous medium, such as a human body, is determined by the hydrophilic fraction (f _phil ) of the amphiphilic copolymer (f _phil = M _{w, phil} / (M _{w, phil} + M _{w, phob} )). As used herein, M _{w, phil} and M _{w, phob} are weighted average molecular weights of the hydrophilic and hydrophobic polymer fractions, respectively. Under aqueous conditions, polymersomes (i.e., vesicles with copolymer blocks) are formed at 0.2 <f _phil <0.4, while polymer micelles are determined at f _phil > 0.5. In the case of polymers, amphiphilic block copolymers are assembled into a bilayer structure of the head-tail and tail-head type.

The self-aggregating structure of the invention may also be a polymer-stabilized oil-in-water emulsion. In this case, a monolayer of amphiphilic polymer is formed around the oil droplet, the hydrophobic part is directed to the surface of the oil, and the hydrophilic part is directed to the surrounding aqueous phase. This finds application, for example, in CT (using iodinated oil), functional MRI (using perfluorinated oil) and drug delivery (there are several emulsions used for drug delivery approved by the FDA, for example, based on soybean oil).

Various details according to the invention are described later in this document.

Amphiphilic polymer

Amphiphilic polymers capable of self-aggregation are known per se, as are the resulting nanocarriers, such as self-aggregated polymer structures. The person skilled in the art has the appropriate laboratory equipment to obtain these polymers. References included in these patent documents, such as WO 2005/016259, US 6835394, US 2005/180922, EP 1279682, US 2008/166382, WO 2008/58963, as well as various minor references are referred to in these documents.

The polymer according to the invention typically comprises at least one end hydrophilic block (A) and at least one end hydrophobic block (B). In a preferred, simplest form, the polymer is a block copolymer having only the two above blocks, i.e. polymer with a general structure AB. These blocks per se preferably mainly consist of single repeating monomer units (M _A , M _B, respectively). The resulting block copolymer structure thus satisfies the general structural formula (i).

where X is a chelating moiety; M _A is a repeating hydrophilic unit; M _B is a repeating hydrophobic unit; n and m each independently are integers representing the number of monomer units forming a block. With regard to the number of repeating units, they should be enough to increase the amphiphilicity of the polymer and should be, as a rule, at least 3. The maximum number, in particular, is not critical, and is determined by the standard estimate associated with the method of producing the polymer. Thus, the standard upper limit is 1,000,000. A preferred range for n and m is from 4 to 40,000, preferably from 5 to 5000, and most preferably from 10 to 225.

However, it is possible that either or both of the hydrophilic and hydrophobic blocks contain two or more different repeating units, thus providing a polymer that satisfies general formula (ii):

In this document, M _A1 , M _A2 and M _A3 denote various hydrophilic repeating units, and M _B1 , M _B2 and M _B3 denote various hydrophobic repeating units. The letters p, q, r, x, y and z each independently represents an integer from 0 to 1,000,000 with the condition that (p + q + r) and (x + y + z) are in the range from 3 to 1,000,000, preferably from 4 to 40,000, more preferably from 5 to 5000, and most preferably from 10 to 225. In similar multiblock polymers, a large number of different repeating hydrophilic and hydrophobic units may be present, however, is not preferred.

Also in the polymers according to any of the above formulas (1) and (2), it is possible to include block (C), a solvent with amphiphilic properties, i.e. a block that is neither hydrophilic nor hydrophobic. The hydrophilic block or blocks are typically water soluble blocks, and are preferably selected from the group consisting of polyethylene oxide, polymethacrylic acid, polyacrylamide derivatives, polyhydric alcohols such as polyvinyl alcohol or polyhydroxyethyl methacrylate, hydrophilic polypeptides and sugar derivatives. Most preferably, the hydrophilic block is a block of polyethylene oxide (PEO, PEG), since the polymer according to the invention is essentially "PEGylated" in the aqueous environment of a human or animal organism, a hydrophilic block of polyethylene oxide, i.e. PEG will form the outer surface of a self-aggregated structure (such as a polymersome), thus providing 100% PEGylation of the surface, and thus optimal stealth (leading to longer circulation times as a result of less opsonization). With reference to the aforementioned fraction f _phil , it is preferable that the weight average molecular weight of the polyethylene oxide block is from 500 to 10,000. Longer hydrophilic blocks will necessitate sufficiently long hydrophobic blocks that are less desirable due to lower biodegradability and more complex process production (high viscosity). As a rule, a hydrophobic block or blocks lacks affinity for water, and they are preferably selected from polymers with Tg below 70 ° C, such as polybutadiene, polyisoprene, polyethylene. Basically, all polymers with a skeleton of carbon atoms and side groups of a hydrophobic nature can be used as a hydrophobic block.

The aforementioned preferred Tg is also, inter alia, related to the production method, since polymers with a Tg greater than the Tg value are more difficult to obtain due to their high viscosity and / or because of their degree of crystallization under the production conditions. In the case of high Tg polymers, it is preferable to use plasticizers during the preparation process (for example, organic solvents such as THF (tetrahydrofuran) or dichloromethylene. These plasticizers are used as a preparation method and removed before using the polymer. This preparation method is usually available to a person skilled in the art polymer science.

As one skilled in the art understands, a large degree of design flexibility is achieved by standard methods of influencing the molecular weight and molecular weight distribution of amphiphilic polymers. This can also be considered in terms of the number of end groups per weight of the polymer unit, which thus provides an easy way to vary the number of chelate groups per weight of the polymer unit. A specific advantage can be achieved by combining a chelating amphiphilic polymer with a sufficiently short hydrophilic chain (namely, a PEG chain) with an amphiphilic polymer with a sufficiently large PEG chain. As a result of self-aggregation, the chelating agent will thus be contained within the polymer layer forming the self-aggregating structure, while PEG chains will form the outer surface of the structure, thus providing the entire surface with PEG, leaving it untouched by the chelating fragments.

Chelating moiety

A chelating moiety can be prepared and / or selected from moieties that contain atoms that are electron donors. These fragments can be selected, for example, from polyphosphates such as sodium tripolyphosphate and hexametaphosphoric acid; aminocarboxylic acids such as ethylenediaminetetraacetic acid, N- (2-hydroxyethyl) ethylenediamine triacetic acid, nitrilotreacetic acid, N, N-di (2-hydroxyethyl) glycine, ethylene bis (hydroxyphenyl glycine) and diethylene triamine pentaacetic acid; 1,3-diketones such as acetylacetone, trifluoroacetylacetone and thienoyl trifluoroacetone; and hydroxycarboxylic acids such as tartaric acid, mucic acid, citric acid, gluconic acid and 5-sulfosalicylic acid; polyamines such as ethylenediamine, diethylenetriamine, triethylenetetramine and triaminotriethylamine; amino alcohols such as triethanolamine and N- (2-hydroxyethyl) ethylenediamine; aromatic heterocyclic bases such as 2,2'-dipyridyl, 2,2'-diimidazole, dipicolinic acid amine and 1,10-phenanthroline; phenols such as salicylaldehyde, disulfopyrocatechol and chromotropic acid; aminophenols such as 8-hydroxyquinoline and oxinsulfonic acid; oximes such as dimethylglyoxime and salicylaldoxime; peptides containing proximal chelate groups, such as polycysteine, polyhistidine, polyaspartic acid, polyglutamic acid or combinations of such amino acids, each polyamino acid contains from 2 to about 20 amino acids in the polymer; Schiff bases such as 1,2-propylenediimine disalicylaldehyde; tetrapyrroles such as tetraphenylporfin and phthalocyanine; sulfur compounds such as toluene dithiol, meso-2,3-dimercaptosuccinic acid, dimercaptopropanol, thioglycolic acid, potassium ethyl xanthate, sodium diethyl dithiocarbamate, dithizone, diethyl dithiophosphoric acid and thiourea; synthetic macrocyclic compounds such as dibenzo-18-crown-6, (CH3) 6 - [14] -4,11-diene-N4 and (2.2.2) -cryptate, and phosphonic acids such as nitrilotrimethylene phosphonic acid, ethylene diamine tetra ( methylene phosphonic acid) and hydroxyethylidene diphosphonic acid, or a combination of two or more of the above compounds.

Preferred chelating fragments contain one or more carboxylic acids or carboxyl groups and include elements present in: ethylenediamine-N, N, N ', N'-tetraacetic acid (EDTA); N, N, N ', N ", N" -Diethylleptriaminepentaacetic acid (DTPA); 1,4,7,10-tetraazacyclododecane-N, N ', N ", N'" - tetraacetic acid (DOTA); 1,4,7,10-tetraazacyclododecane-N, N ', N "-acetylacetoacetic acid (DO3A); l-oxa-4,7,10-triazacyclododecane-N, N', N" -acetylacetoacetic acid (OTTA); trans- (l, 2) -cyclohexanodiethylenetriamine pentaacetic acid (CDTPA).

The most preferred chelating fragments are DOTA, DTPA, HYNIC (6-hydrazinonicotine used to chelate technetium) and desferoxamine (for example, available as desferoxamine mesylate under the patented name Desferal), used for chelation with gallium.

One skilled in the art will understand that the above examples are chelating compounds and that chelating fragments, when compared with these compounds, are essentially derivatives thereof in the sense that they include a bond with the polymer.

Linking chelating fragment

Despite the fact that other production sequences are not excluded, it is preferable to first obtain an amphiphilic copolymer and then attach a chelating fragment to its hydrophilic block. One skilled in the art will recognize that the strict binding between the hydrophilic end monomer and the chelating moiety is determined by the functional groups available at the end of the monomer and on the chelating moiety which serves to form the chelating moiety in the polymer. The standard, most commonly used type of bond is an amide bond.

A preferred block of poly (butadiene) poly (ethylene oxide) polymers according to the invention can be prepared as follows, as shown in Scheme 1 below. First, a polymer block is provided, in this case poly (ethylene oxide) -block poly (butadiene) (1). The primary alcohol of the polymer is converted to the corresponding tosylate of the copolymer (2). Then, the tosylate is reacted with NH ₃ to obtain an amino functionalized poly (ethylene oxide) block poly (butadiene) (3). Then, the amino group of Ponte 3 is reacted with DOTA N-succinimidyl ether (4) to obtain DOTA-functionalized poly (ethylene oxide) -block-poly (butadiene) (5).

Scheme 1

Herein, reaction compounds and solvents are listed below: (i) pTsCl, KOH, DCM; (ii) 7 N NH ₃ , toluene / MeOH; (iii) Et ₃ N, DMF.

Depending on the application of interest, the DOTA functionalized block copolymer (5) can be mixed with non-functionalized block copolymers in the ratio between a minimum of one chelate polymer to a maximum of 100% in the system (i.e. all polymer molecules present are chelate polymers). The developed synthesis method can be used for a wide range of copolymers with different molecular weights and various f _phil values, which allows to obtain a wide range of self-aggregated structures. In addition, a similar synthesis strategy can be used to chemically modify block copolymers with other metal-related ligands, such as DTPA, and target ligands, such as antibodies, peptides, etc.

Coordination complexes based

chelating fragments

The chelating compounds represented in the amphiphilic polymer according to the invention can be used to create coordination complexes with metals in accordance with the chelation methods known in the art.

Since the amphiphilic polymers according to the invention are essentially provided with coordination sites, there is extensive freedom of choice with respect to the degree of chelation, which can range from one chelating ion to the maximum achievable (with the possibility of achieving a much higher degree of chelation than with standard polymers, which per se do not form chelating compounds).

Using an example of a copolymer based on DOTA, as described above, in one of the methods, this polymer is capable of self-aggregation into polymersomes, with subsequent formation of a complex with Gd (III). In the second method, the reaction with Gd (III) and a DOTA-based copolymer is carried out in a first step and, subsequently, the Gd (III) DOTA-functionalized copolymers self-aggregate into polymersomes. In the first method, Gd (III) is present only on the outer surface of the polymersome, i.e. complexes with Gd (III) are directed outward into the water column, while in the second method, complexes with Gd (III) are directed outward on both sides of the polymersome membrane.

Contrast for T _one / T ₂  MRI

The polymers according to the invention can be used as contrast agents for MRI. As a rule, this applies to suspended contrast agents for T ₁ and / or T ₂ .

In a magnetic resonance imaging study of a mammalian organism, such as a human, an in vivo image of an organ or tissue is obtained by placing at least a part of the body to be imaged in a strong external magnetic field excited by the energy of the radio emission, and observe the effect of the excitation on the magnetic properties photons contained in the organ or tissue and surrounding them. This is especially useful when visualizing the blood flow of an organism (i.e., the bloodstream). A number of magnetic parameters can be measured. The proton relaxation time, T ₁ and T ₂ , are paramount. T ₁ , also called spin-lattice, or the period of longitudinal relaxation, and T ₂ , also called spin-spin, or the period of transverse relaxation, are functions of the chemical and physical aqueous environment of the internal environment of an organ or tissue, and they are measured using radio pulse methods. This information is analyzed as a function of spatial location using a computer that converts information to obtain an image.

Often, the resulting images lack adequate contrast, for example, between normal and damaged tissue, which leads to a decrease in the efficiency of diagnosis. To overcome this drawback, contrast agents are used. Magnetic resonance contrast agents are magnetically active compounds that affect the magnetic resonance parameters of the nuclei of molecules that are close to them. Theoretically, a contrast agent, if it is preferable to consider a specific part of an organ or a specific type of tissue, for example, affected tissue, can provide a change or enhance the contrast of the resulting image of the given tissue.

Since the magnetic resonance image is strongly influenced by changes in the parameters T ₁ and T ₂ , it is desirable to have a contrast medium that affects either of two or both parameters. Studies are concentrated mainly in relation to two classes of magnetically active materials, i.e. paramagnetic materials that lower T ₁ and T ₂ ; and superparamagnetic materials that mainly lower T ₂ . At low concentrations, paramagnetic materials have a greater effect on T ₁ than on T2.

Paramagnetism occurs in materials containing electrons with unpaired spins. Paramagnetic materials are characterized by weak magnetic susceptibility (response to an external magnetic field). Paramagnetic materials become weakly magnetic in the presence of a magnetic field and quickly lose this type of activity when the external field is removed, i.e. demagnetized. For a long time it was found that the addition of paramagnetic materials to water causes a decrease in the T ₁ parameters of hydrogen nuclei.

As contrast agents for MRI, paramagnetic materials containing, for example, paramagnetic lanthanides, especially materials containing Gd ⁺³ , are preferred, primarily due to their effect on T ₁ .

Due to the presence of a chelating moiety in the amphiphilic polymers of the invention, the paramagnetic material can simply be incorporated into the polymer, allowing it to form a coordination complex with the chelating moiety.

In the case of contrast agents for T ₁ / T ₂ , it is preferable if they are based on self-aggregating structures with paramagnetic material present on the outer surface. In this case, micelles are thus suitable. However, it is preferable that the nanocarrier according to the invention be in the form of a polymersome.

SPECT and PET contrast agents

Similarly, as in the case of the formation of coordination complexes with paramagnets, the polymers according to the invention can also be used to include radionuclides.

With single-photon emission computed tomography (SPECT), images are obtained that reflect the distribution of gamma radiation released by nuclides. This method of image acquisition has a very high sensitivity and the absence of a background signal, allows to obtain quantitative data on the biodistribution of the radionuclide. SPECT is usually used in a hospital setting to visualize and quantify areas of the tumor and then to assess the biodistribution of potentially new drugs or contrast agents. Recent developments include the synthesis of lipid-stabilized SPECT emulsions using ¹¹¹ In as a radionuclide. The incorporation of DTPA or DOTA functionalized copolymers into polymersomes, polymer micelles, and copolymer-stabilized emulsions allows for the efficient radioactive labeling of self-aggregated particles using radioactive isotopes (such as ¹⁷⁷ Lu or ¹¹¹ In) for use in the field of radionuclide imaging.

The present invention includes a method for producing and radioactively labeling a polymer-stabilized emulsion using an amphiphilic polymer, as previously described herein, for example, using DOTA-functionalized poly (ethylene oxide) -block-poly (butadiene) as an emulsifier.

As evidence of the concept, emulsions were stabilized using a copolymer prepared by the present inventors loaded with DOTA, and these structures were labeled with a radioactive isotope using ¹¹¹ In. Biodistribution of compounds was studied in mice. In FIG. Figure 7 SPECT / CT scan shows ¹¹¹ In presence in the heart, liver, and kidneys. The presence of ¹¹¹ In in the blood 4 hours after administration is a sign of a long circulation time in the blood of emulsions labeled with a radioactive isotope. In addition, accumulation in the liver reflects the excretion of nanoparticles by the hepatobiliary route, which was later confirmed by the absence of ¹¹¹ India in the bladder. The results show that emulsions labeled with a radioactive isotope using the DOTA copolymer prepared by the present inventors can be used as a contrast agent for SPECT.

Similarly, the present invention finds application in PET, providing chelation with radionuclides, typically used in PET, such as Rubidium-82, Gallium-68, Copper-64 and Zirconium-89.

Contrast media for CEST MRI

The polymers according to the invention, self-aggregated in polymersomes, are a suitable basis for obtaining a contrast agent for CEST MRI. The CEST method is used to contrast the image using saturation transfer depending on chemical exchange (CEST) from individual pre-magnetically saturated protons to molecules of the water column determined by MRI.

CEST in combination with paramagnetic chemical shift reagents (ParaCEST) is a method in which the magnetization of a pool of protons, the chemical shift of which is caused by paramagnets, of the contrast agent for CEST is selectively saturated using high-frequency radiation (RF). The transfer of this saturation to the molecules of the water column through proton exchange leads to a decrease in the number of excited water protons surrounded by the CEST contrast medium. Thus, a decrease in the signal intensity of molecules of the water column is observed, which can be used to create (neutralize) the enhancement of contrast in MRI images.

The approach to obtaining high efficiency in CEST is based on the use of a large number of water molecules from a solution containing paramagnetic reagents for shear, for example, Na [Tm (dotma) (H ₂ O)]), where "H ₄ dotma" is a (lpha) , a ', a, "a'" is tetramethyl-1,4,7,19-tetraacetic acid, and dotma is the corresponding fourfold deprotonated tetra anionic form of the ligand to provide a pool of protons that are chemically sheared and thus can be selectively saturated with an RF pulse. If this system is encapsulated in a carrier, in this case, in polymersomes, magnetic saturation can be transferred to molecules in the water column outside the carrier that are not subjected to chemical shift. The amount of transfer of magnetization and, thus, the degree of enhancement of contrast, is determined using the coefficient of diffusion of water through the shell of the carrier (i.e. the coefficient of exchange of water), as well as using the amount of water inside the carrier.

The optimal water exchange coefficient is strictly correlated with the difference in chemical shift between the proton pool inside the carrier and the water column outside the carrier. Two main processes contribute to the paramagnetic shift induced on water molecules inside the polymers: the chemical shift caused by the direct interaction of dipoles between water molecules and the shift reagent (δ _dip ), and the chemical shift caused by the volumetric effect of magnetic susceptibility (δ _bms ) The total paramagnetic shift is the sum of the above processes:

δ _bms for spherical particles is zero, but for anisotropic particles it can be significant. Aspherical particles experience an increase in the magnetic field, which causes them to line up in line with the lines of force of the magnetic field. In the case of liposomes, it was shown that the total paramagnetic shift can be further increased if paramagnetic molecules associated with the phospholipid membrane are created.

A reference to the use of aspherical liposomes for CEST is Terreno, E. et al. Angew. Chem. Int. Ed. 46, 966-968 (2007).

Due to the presence of a chelating moiety in the amphiphilic polymer of the invention, the paramagnetic material can be easily incorporated into the polymer, allowing it to form a coordination complex with the chelating moiety. This facilitates incorporation into the polymer of a suitable paramagnetic material (preferably lanthanide, and most preferably Tm or Dy) in any ratio.

Similar to the above-described method for producing contrast agents for MRI (T ₁ / T ₂ ), lanthanides can be provided on the outer surface or also on the inner surface of the polymersome. It should be noted that with respect to CEST, the invention is successful in terms of design freedom. Polymersomes can be used as a more or less standard contrast agent for CEST, providing a pool of protons inside the cavity with a suitable paramagnet, such as lanthanide in liquid form or in suspension. The external chelating surface of the polymersome can be used to create a coordination complex with an additional paramagnetic metal, whereby the difference in the chemical shift for transferring the saturation of protons in the medium by the polymers can be increased (thus, the above effect of magnetic susceptibility is enhanced). Additionally, paramagnetic materials within the cavity can be provided in the form of a coordination complex with chelating fragments in the polymer according to the invention.

In addition, in any of the above embodiments, the polymersomes can be aspherical to enhance the CEST effect. Polymersomes are usually spherical. Giving the polymersomes an aspherical shape is carried out by subjecting them to a dialysis process against a hypertonic buffer solution, that is, a buffer solution with a higher osmolality compared to the solution inside the polymers. Dialysis is the cause of the pure diffusion of water from the interior by polymers into the bulk of the solution. This reduces the total amount of internal space by polymers. Since the surface of the polymers remains unchanged, a decrease in volume causes the polymersomes to deform, to take an aspherical shape, such as the shape of a disk, cigar, or any other aspherical shape.

It should be noted that, in the case of aspherical polymers, the CEST effect can also be fully achieved based on the effect of the non-spherical shape, i.e. without a reagent with a paramagnetic shift, mainly interacting with the material analyzed by MR present in the cavity, or with material selected for analysis by MR, which lacks interaction with a reagent with a paramagnetic shift present in the cavity. In the present invention, this provides additional design flexibility: as indicated above, a cavity can be selected so that it is not filled with metal. In this case, polymersomes provided in aspherical form can be used as contrast agents for CEST, however, to introduce additional contrast, for example, based on T ₁ and / or T ₂ , chelation with the participation of metals on the outer surface can be used.

The CEST effect can be further adjusted using the natural properties of the copolymer blocks and / or the density of the polymer layer, since these parameters affect the coefficient of water exchange through the membrane; for example, the amphiphilic nature of the polymer can be used to influence the coefficient of proton exchange through polymersomes. This, as a rule, can be done by changing the ratio of the lengths of more hydrophilic and more hydrophobic blocks.

With regard to water exchange, it is understood that the CEST effect can also be achieved in the case of other materials analyzed by MR, such as small organic molecules, as well as molecules capable of exchange through a membrane bilayer polymersome.

Drug delivery

Various diseases, which are mainly localized in a particular tissue, are treated by systemic administration of drugs. A well-known example of standard antitumor therapy is systemic chemotherapy, accompanied by significant side effects for the patient due to undesirable biodistribution and toxicity. The therapeutic window for these drugs, on the one hand, is usually determined by the minimum necessary therapeutic concentration in the affected tissue, and, on the other hand, by the types of toxic effect on organs that are not targets, for example, the liver, spleen. Local exposure, for example, through the local release of cytostatics from nanocarriers, promises a more effective treatment and a wider therapeutic window compared to standard treatment. Local drug delivery is also important if other types of therapeutic effects, such as surgical ones, are too dangerous, as is often the case with malignant liver tumors. Local drug delivery may also be the preferred treatment for many indications for cardiovascular disease (CVD), such as coronary arteriosclerosis.

Magnetic resonance imaging is an important diagnostic method, commonly used in a hospital for the diagnosis of diseases. MRI allows for non-invasive imaging of soft tissues with excellent spatial resolution.

As a useful extension of diagnostic applications, it is also proposed to use MRI to monitor the delivery of biologically active agents, such as therapeutic or diagnostic compounds. Those. MRI can be used not only for treatment planning, but also for monitoring local drug delivery with visual control. A reference to this is Ponce et al., J Natl Cancer Inst 2007; 99: 53-63. In this document, the drug, doxorubicin, is enclosed in a temperature-sensitive liposome, which is in the solid state at normal body temperature, and melts when the temperature increases by several degrees (41-42 ° C). Thus, the release of the drug can be facilitated by the application of heat, which will result in the opening of the liposome, whereby the release of the drug is no longer determined by diffusion (if provided) through the liposome membrane. To monitor the release of the drug using MRI, manganese salts are added to the drugs used as the contrast medium.

The polymersomes according to the invention can be used as a carrier for a drug. The administration and delivery of a drug introduced into the body using such a carrier can be monitored using T ₁ / T ₂ and / or CEST MRI, depending on (as understood and described above, based on the present invention) the type and location of paramagnetic metal complexed with amphiphilic chelate polymer.

The drug carrier is to be introduced into the body of a person undergoing MRI. By, for example, injection into the bloodstream, or other methods of introducing a carrier into a body fluid.

A medicinal product is a chemical compound used to treat, treat, prevent or diagnose a disease or disorder, or otherwise used to improve physical or mental well-being. The controlled delivery contemplated by the present invention is generally effective with respect to therapeutic agents (i.e., in the strict sense, drugs intended for the treatment or prophylaxis of diseases or disorders), but also with respect to compounds administered for diagnostic purposes. Although, other biologically active agents, i.e. non-therapeutic or diagnostic agents, such as food products, are not usually delivered in a controlled and / or controlled manner, but if desired, this can be done using the present invention.

The most optimal method of use according to the invention is achieved in the case of a targeted therapeutic effect, i.e. in the case of drugs intended for targeted delivery, since such delivery by nature has the greatest benefit from monitoring available through the invention. This applies, for example, to compounds with local delivery in the treatment of tumors, to compounds for the treatment or prophylaxis of cardiovascular diseases such as coronary arteriosclerosis, or to antithrombotic compounds (for example, to locally dissolve blood clots) or compounds that need to be crossed blood-brain barrier, such as neuromodulators, which can be used in the treatment of neurological diseases such as epilepsy, Alzheimer's disease, Parkinson's disease or stroke. The benefits of managing and monitoring the delivery of targeted drugs are also suitable for targeted connections for diagnostic purposes. As with targeted therapy, here, too, tumors are an area where site-specific delivery is extremely important.

Biologically active agents suitable for use in the present invention include biologically active agents, including therapeutic drugs, endogenous molecules and pharmacologically active agents, including antibodies; molecules intended for nutrition; compounds for diagnostic purposes; and additional contrast agents for visualization. As used herein, the active agent includes pharmacologically acceptable salts of the active agents.

The drug carriers of the present invention based on polymersomes may contain both hydrophilic and hydrophobic biologically active agents. A hydrophilic biologically active agent may be included in the aqueous compartment of the carrier or may be associated with a more hydrophilic part of the particle shell, or its distribution may include a combination of these possibilities, while hydrophobic biologically active agents may be included in the hydrophobic domains of the carrier, for example, in the polymer shell . Nucleic acids, carbohydrates, and mainly proteins and peptides are water soluble or hydrophilic. For example, biologically active agents that are low molecular weight compounds, lipids, lipopolysaccharides, polynucleotides and antisense nucleotides (compounds for gene therapy) can also be considered. Biologically active agents that may be included, thus, include drugs that are not peptides and proteins. Within the framework of the present invention, it is possible to include drugs of polymer nature, but also to include drugs with a sufficiently small molecular weight of less than 1500 g / mol or even less than 500 g / mol.

Thus, compounds contemplated for use as biologically active agents in the context of the present invention include any compounds with therapeutic or prophylactic properties. It can be a compound that affects or participates in tissue growth, cell growth, cell differentiation, a compound that can cause a biological effect, such as an immune response, or a compound that can play any other role in one or more biological processes. A non-limiting list of examples includes antimicrobial agents (including antibacterial, antiviral and antifungal agents), antiviral agents, antitumor agents, thrombin inhibitors, antithrombotic agents, thrombolytic agents, fibrinolytic agents, angiospasm inhibitors, calcium channel blockers, anti-vasodilators , antibiotics, surface glycoprotein receptor inhibitors, antiplatelet agents agents, antimitotic agents, microtubule inhibitors, antisecretory agents, actin inhibitors, remodeling inhibitors, antimetabolic agents, including angiogenesis inhibitors, anticancer chemotherapeutic agents, anti-inflammatory steroid or non-steroidal anti-inflammatory drugs, growth hormone anti-inflammatory drugs, anti-inflammatory drugs dopamine, radiotherapy agents, extracellular matrix components, ACE inhibitors, free radical traps, chelating compounds, antioxidants, polymerase inhibitors and phototherapy agents.

Mention may be made of rather small peptides in terms of the number of amino acids (e.g. di-, tri-, tetrapeptides). A peptide with a sufficiently small number of amide bonds can also be called an oligopeptide (up to 50 amino acids), while a peptide with a sufficiently large number (more than 50 amino acids) can be called a polypeptide or protein. In addition to the existence of a polymer with amino acid residues, some proteins, in addition, can be characterized by the so-called quaternary structure, a combination of several polypeptides, which are not necessarily chemically linked by amide bonds, but bound by the interaction forces, usually known to specialists in this field, such as electrostatic forces and van der Waals forces. The term peptides, proteins, or mixtures thereof, as used herein, includes all of the above possibilities.

Typically, a protein and / or peptide is selected based on their biological activity. Depending on the type of polymer selected, the product obtained by the present method is extremely suitable for the controlled release of proteins and peptides. In a specific embodiment, the protein or peptide is a growth factor.

Other examples of peptides or proteins or compounds containing peptides or proteins that can be successfully incorporated into a loaded polymer include, but are not limited to, immunogenic peptides or immunogenic proteins, which include, but are not limited to, the following:

Toxins such as diphtheria toxin and tetanus toxin.

Surface antigens of a virus or parts of viruses, such as adenoviruses, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, herpes viruses, HIV-1, HIV-2, human T-cell leukemia virus-III, influenza viruses, Japanese encephalitis virus , measles virus, papilloma virus, paramyxoviruses, poliomyelitis virus, rabies virus, rubella virus, vaccinia viruses (smallpox) and yellow fever virus.

Bacterial surface antigens or parts of tacteria, such as Bordetella pertussis, Helicobacter pylori, Clostridium tetani, Corynebacterium diphtheria, Escherichia coli, Haemophilus influenza, Klebsiella species, Legionella pneumophila, Mycoberium tuberiumerium, Mycoberium bacteria, Mycobereria tuberium levella, Mycoberium tuberium levella , Pseudomonas aeruginosa, Salmonella species, Shigella species, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholera and Yersinia pestis.

Superficial antigens of pathogenic parasites or parts of parasites, such as Plasmodium vivax (malaria), Plasmodium falciparum (malaria), Plasmodium ovale (malaria), Plasmodium malariae (malaria), Leishmania tropica (leishmaniasis), Leishmanislimis doni ), Trypanosoma rhodescense (sleeping sickness), Trypanosoma gambiense (sleeping sickness), Trypanosoma cruzi (Chagas disease), Schistosoma mansoni (schistomatosis), Schistosomoma haematobium (schistomyiasis), Schistosoma japonicum (schistomyosis), trichinella spiralis (Trichinella spiralis) hookworm), Ancyclostoma duodenale (hookworm), Necator americanus (hookworm), Wucheria bancrofti (filariasis), Brugia malaya (filariato ), Loa (filariasis), Dipetalonema perstaris (filariasis), Dracuncula medinensis (filariasis), and Onchocerca volvulus (filariasis).

Immunoglobulins such as IgG, IgA, IgM, rabies immunoglobulins, and vaccinia virus immunoglobulins.

Anatoxins such as botulinum toxoid, diphtheria toxoid, gangrenous toxoid, tetanus toxoid.

Antigens that trigger an immune response against foot and mouth disease.

Hormones and growth factors, such as follicle-stimulating hormone, prolactin, angiogenin, epidermal growth factor, calciotonin, erythropoietin, thyrotropic releasing hormone, insulin, insulin-like growth factor 1 and 2, bone growth factor, human chorionic gonadotropin hormone, luteinizing nerves, adrenocorticotropic hormone (ACTH), releasing luteinizing hormone factor (RF-LH), parathyroid hormone (PTH), thyrotropin-releasing hormone (TRH), vasopressin, cholecystokenin and corticoliberin; cytokines such as interferon, interleukins, colony stimulating factor and tumor necrosis factor: fibrinolytic enzymes such as urokinase, renal plasminogen activator; coagulation factors such as protein C, factor VIII, factor IX, factor VII and antithrombin III.

Examples of other proteins and peptides are albumin, atrial natriuretic factor, renin, superoxide dismutase, alpha 1-antitrypsin, pulmonary surfactant proteins, bacitracin, bestatin, cidosporin, delta-son-inducing peptide (PVDS), endorphins, glucagon, gramotinin, gramicinin, gramicinin, gramicinin, gramicinin factor, neurotensin, oxytoin, somatostatin, terrototide (terprotide), thymic factor of the serum, thymosin, desmopressin, dermorphin, met-enkephalin, peptidoglycan, sateitin, thymopeptin, fibrin decay products, desenkefalin-alpha-endorphin gonadolin, n, leuprolide, MSH-alpha and metcofamide.

Antineoplastic agents, such as altretamine, fluorouracil, amsacrine, hydroxycarbamide, asparaginase, ifosfamide, bleomycin, lomustine, busulfan, melphalan, chlorambucil, mercaptopurine, chloromethine, methotrexate, cisplatin tibidazbitazbitazidazbitazidazbitazidazbitazidazitbinazidinozibitazidinozibitazidinitazbitazitazidinitazbitazitazidinozibitazidinozibitazidinozibidazbittibitazidinitazbitin , thioguanine, daunorubicin, threosulfan, doxorubicin, thiophosphamide, estramustine, vinblastine, etoglucid, vincristine, etoposide, vindesine and paclitaxel.

Antimicrobial agents contain:

Antibiotics, such as ampicillin, nafcillin, amoxicillin, oxacillin, azlocillin, penicillin G, carbenicillin, penicillin V, dicloxacillin, phenethillin, phloxacillin, piperacillin, mecillin, sulbenicillin, ticillicillin, methicillin, methicillin, methicillin, methicillin, methicillin, methicillin, methicillin. Cephalosporins: cefaclor, cephalotin, cefadroxil, cefapirin, cefamandole, cefradin, cefatrizin, cefsulodin, cefazolin, ceftazidime, ceforanide, ceftriaxone, cefoxitin, cefuroxime, cefcefil, cefcefil. Aminoglycosides, such as amikacin, neomycin, dibecacin, kanamycin, gentamicin, netilmicin, tobramycin. Macrolides, such as amphotericin B, novobiocin, bacitracin, nystatin, clindamycin, polymyxin, colistin, rovamycin, erythromycin, spectinomycin, lincomycin, vancomycin. Tetracyclines such as chlortetracycline, oxytetracycline, demeclocilin, roletetracycline, doxycycline, tetracycline and minocycline. Other antibiotics such as chloramphenicol, rifamycin, rifampicin and tiamphenicol.

Chemotherapeutic agents, such as sulfonamides sulfadiazine, sulfamethizole, sulfadimethoxine, sulfamethoxazole, sulfadimidine, sulfamethoxypyridazine, sulfafurazole, sulfafenazole, sulfalene, sulfisomidine, sulfamerazine, sulfisoxazole and trimethoprim with sulfamethoxazole or sulfamethrolamine.

Uroseptics, such as methylamine, quinolones (norfloxacin, cinoxacin), nalidixic acid, nitro compounds (nitrofurantoin, nifurtoinol) and oxolinic acid.

Medicines against anaerobic infections such as metronidazole.

Medicines for treating tuberculosis such as aminosallicylic acid, isoniazid, cycloserine, rifampicin, ethambutol, thiocarlide, ethionamide and viomycin.

Medicines for the treatment of leprosy, such as amityozone, rifampicin, clofazimine, sodium sulfoxone and diaminodiphenylsulfone (DDS, dapsone).

Antifungal drugs such as amphotericin B, ketoconazole, clotrimazole, miconazole, econazole, natamycin, flucytosine, nystatin and griseofulvin.

Antiviral drugs such as acyclovir, idoxuridine, amantadine, metisazon, cytarabine, vidarabine and ganciclovir.

Medicines for the treatment of amoebic dysentery, such as chloroquine, iodokinol, clioquinol, metronidazole, dehydroemethine, paromomycin, diloxanide, furoatetinidazole and emethine.

Antimalarial drugs such as chloroquine, pyrimethamine, hydroxychloroquine, quinine, mefloquine, sulfadoxine / pyrimethamine, pentamidine, sodium suramine, primaquine, trimethoprim and proguanil.

Anthelmintic drugs, such as antimonyl potassium salt of tartaric acid, niridazole, antimony sodium dimercaptosuccinate, oxamnichine, befenium, piperazine, dichlorofen, praziquantel, diethylcarbamazine, pyrantel pamoate, gikmofolzenol pyrtolomene pyrtolum tetrazolimetol pyrtolumtibenzolimetol pyrtolum

Anti-inflammatory drugs such as acetylsallicylic acid, mefenamic acid, aclofenac, naproxen, azopropanone, niflumic acid, benzidamine, oxyphenbutazone, diclofenac, piroxicam, phenoprofen, pyrrofrofen, flurbiprofen, salicrofenophenolticene, indicrofin, tacinoferfin indolithrofen, tricyrofen indolithrofen, tricyrofen indolithrofen, tricyrofen indolithrofen, tricyrofen indofin, .

Anti-gout medications such as colchicine and allopurinol.

Central-action analgesics (opioids) such as alfentanil, methadone, bezitramide, morphine, buprenorphine, nicomorphine, butorphanol, pentazocine, codeine, pethidine, dextromoramide, pyritramide, dextroproxifen, sufentanil and fentanyl.

Local anesthetics such as articaine, mepivacaine, bupivacaine, prilocaine, ethidocaine, procaine, lidocaine, and tetracaine.

Medicines for the treatment of Parkinson's disease, such as amantadine, diphenhydramine, apomorphine, etopropazine, benztropine mesylate, lergotril, biperiden, levodopa, bromocriptine, lysuride, carbidopa, methixene, chlorphenoxamine, orphenadrine, cyclimine, proxytimenidine and dimexidilidine.

Centrally acting muscle relaxants such as baclofen, carisoprodol, chlormezanone, chlorozacsozone, cyclobenzaprine, dantrolene, diazepam, febarbamate, mefonoxalon, mefenesin, metaxalone, methocarbamol and tolperisone.

Corticosteroids contain:

Mineralcorticoids such as cortisol, deoxycorticosterone and fluorohydrocortisone.

Glucocorticosteroids contain beclomethasone, betamethasone, cortisone, dexamethasone, fluocinolone, fluocinonide, fluocortolone, fluorometholone, fluprednisolone, flurandrenolide, galcinonide, hydrocortisone, medrizone, methylprednisolone prednisone, parametrizone, prednisone, acetone.

Androgens contain:

Such therapeutic androgenic steroids as danazol, fluoxymesterone, mesterolone, methyltestosterone, testosterone and their salts.

Therapeutic anabolic steroids such as calusterone, nandrolone and their salts, drostanolone, oxandrolone, ethylestrenol, oxymetholone, methandriol, stanozolol, methandrostenolone and testolactone.

Antiandrogens such as cyproterone acetate.

Such therapeutic estrogens, including estrogenic steroids like diethylstilbestrol, estradiol, estriol, ethinyl estradiol, mestranol, hinestrol.

Antiestrogens such as chlorotrianisin, clomiphene, ethamoxytriphenol, nafoxidine and tamoxifen.

Progestins, such as allylestrenol, desogestrel, dimethisterone, dydrogesterone, ethinylestrenol, ethisterone, ethinadiol diacetate, ethinodiol, hydroxyprogesterone, levonorgestrel, linestrenol, medroxyprogesterone, megestrol acetate, norethredestone norethrone donor, norethinestrone, norethinestrone, norethinestrone, norethinestrone, norethinestrone, norethinestrone, norethinestrone

Thyroid hormones include:

Therapeutic thyroid medications such as levothyron and lyothiron.

Therapeutic anti-thyroid medications such as carbimazole, methimazole, methylthiouracil and propylthiouracil.

In addition to biologically active agents that are soluble in water, other water-soluble substances such as antioxidants, ions, chelating agents, colorants, imaging agents can be included.

Preferred therapeutic agents are those for use in tumors (e.g., antitumor) and cardiovascular diseases.

Methods for preparing derivatives of lipophilic drugs suitable for nanoparticle preparations or polymers are known in the art (see, for example, US 5,534,499 for the covalent attachment of therapeutic agents to a phospholipid fatty acid chain). The drugs of the present invention may also be prodrugs.

The drug may be present in the inner, outer or both compartments of the carrier, for example, in the cavity and / or in the shell of the polymersome. The distribution of the drug is independent of the distribution of any other compounds contained in the drug carrier, such as a paramagnetic chemical shift compound or paramagnet. You can use a combination of drugs, and any of these drugs can be present inside, outside or in both compartments of the drug carrier, for example, in the cavity and / or in the shell of the polymersome.

Additional applications:

As noted above, the chelated amphiphilic polymers according to the invention can be combined with non-chelated amphiphilic polymers.

Also, in the case of polymers based on chelate amphiphilic polymers, disease-specific molecules can be provided in the polymersome shell, for example, in the presence of compounds having a hydrophobic tail suitable for penetrating the surface of the polymersome, where the other end of the compound contains the desired ligand. This allows the use of polymersomes as contrast agents, which can preferably localize the desired or suspected areas in the body, which can then be visualized using MRI.

It should be understood that the invention is not limited to the embodiments and formulas as previously described herein. It is also understood that in the claims, the word “comprising” does not exclude other elements or steps. In the case of using the indefinite or definite article, referring to the singular, for example, "a" or "an", "the", this includes the plural of that noun, if, in particular, they do not report anything else.

The present invention is illustrated with reference to the following non-limiting examples and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Nanostructures capable of self-aggregation, containing amphiphilic copolymers. Polymersomes (left), polymer-stabilized emulsions (in the middle), and polymer micelles (right).

FIG. 2. Schematic representation of functionalized with Gd (III) DOTA polymers as T _1,2 -weighted contrast agents for magnetic resonance imaging (MRI).

FIG. 3. Schematic representation of functionalized using DOTA polymers. Spherical polymersomes containing DOTA terminated copolymers in the polymer layer (6, left). As a result of the reaction of DOTA molecules with paramagnetic metals, spherical polymersomes are formed (10, middle), in which paramagnetic complexes are directed outward to the volume of water. Deformation of polymers (10) in response to osmotic pressure makes it possible to obtain aspherical polymersomes (11, right).

FIG. 4. Schematic representation of DOTA-functionalized polymers containing a chemical shift compound in the inland water compartment. Spherical polymersomes containing DOTA terminated copolymers in the polymer layer (12, top left). As a result of the reaction of DOTA 12 molecules with paramagnetic metals, spherical polymersomes are formed (13, top in the middle), in which paramagnetic complexes are directed outward to the volume of water. Using deformation 12 in response to osmotic pressure, aspherical polymersomes can be obtained (15, bottom). Aspherical polymersomes containing compounds with a chemical shift inside and paramagnetic complexes directed outward to the volume of water (14, top right) can be obtained using either 13 or 15.

FIG. 5. Schematic representation of contrast compounds with CEST for MRI, containing paramagnetic complexes of DOTA copolymers on both sides of the polymer layer with polymers. Spherical polymersomes (16, upper left), aspherical polymersomes (17, upper right), spherical polymersomes containing a chemical shift compound in the inland water compartment (18, lower left), and aspherical polymersomes containing the chemical shift compound in the inland water compartment (19, bottom right).

FIG. 6. Polymersomes stabilized by emulsion polymers and polymer micelles for radionuclide imaging, exposing the label, for example, for radionuclide imaging, on the outer surface of the structure.

FIG. 7. SPECT / CT tomogram using an emulsion labeled with ¹¹¹ India 4 hours after injection. The projection with the maximum value of the CT image intensity is recorded together with the SPECT tomogram (top left); coronary SPECT / CT section, which allows you to visualize the heart and liver (top right); Sigittal SPECT / CT section, which allows you to visualize the heart and liver and kidneys (bottom left); transverse SPECT / CT slice (bottom right).

EXAMPLES

Example 1

Synthesis of DOTA-functionalized poly (ethylene oxide) -block poly (butadiene) (5)

PBD (2500) -b-PEO (1300) (1) was dissolved in acetone (18 ml), Yu and the solution was concentrated under reduced pressure to remove excess isopropanol. To remove residual water, the copolymer was dissolved in toluene (15 ml) and this solution was concentrated in vacuo. Subsequently, PBD (2500) -b-PEO (1300) (4.90 g, 1.29 mmol) was dissolved in DCM (15 ml) under nitrogen atmosphere. The resulting solution was cooled to 0 ° C. and p-tosyl chloride (0.497 g, 2.6 mmol) was added. The mixture was stirred for 30 minutes at 0 ° C and KOH (0.640 g, 11.4 mmol) was carefully added. The mixture was stirred overnight at room temperature. The reaction mixture was washed with water (2 × 30 ml) and brine (2 × 15 ml). The aqueous layer was removed using DCM (30 ml), and the mixed organic layers were dried over MgSO ₄ , the filtered contents and the solution were concentrated under reduced pressure to obtain 2 (62%, 3.2 g, 0.81 mmol). The tosylate functionalized copolymer (2) (3.2 g, 0.81 mmol) was dissolved in toluene (12 ml) and a solution of 7 N NH ₃ in MeOH (12 ml, 84 mmol) was added. The reaction was carried out at 50 ° C for 63 hours. Then, the solvent was removed under reduced pressure. The crude mixture was dissolved in DCM (10 ml). The resulting solution was washed with water (2 × 20 ml), brine (2 × 10 ml), and saturated with NaHCO ₃ (aq.) (10 ml). The aqueous layer was removed using DCM (40 ml). The mixed organic layers were dried over MgSO _4. The suspension was filtered and the filtered content was concentrated under reduced pressure to obtain 3 (1.55 g, 0.41 mmol) in 50% yield. The amine functionalized copolymer (3) (1.2 g, 0.31 mmol) was dissolved in DMF (12 ml) and, subsequently, structural blocks based on DOTA (4) (0.347 g, 0.35 mmol) and Et were added ₃ N (0.9 ml, 6.5 mmol). The mixture was stirred for 26 hours at room temperature under nitrogen. The resulting solution was concentrated under reduced pressure. The crude mixture was dissolved in toluene, and the solution was concentrated under reduced pressure. DOTA functionalized poly (ethylene oxide) -block poly (butadiene) (5) was obtained in quantitative yield.

Example 2

Self-aggregation of DOTA-functionalized copolymers and complexation with Gd (III)

Polymer vesicles with an average diameter of 100-150 nm were obtained using the method of hydration of a thin film together with subsequent displacement. Briefly, DOTA functionalized poly (butadiene (l, 2-addition) -b-ethylene oxide) (M _n (g / mol): PBD (2500) -b-PEO (1300), PD = 1.04, and f _EO = 0.34) was dissolved in CHCl ₃ . The solvent was carefully removed under reduced pressure to give a thin polymer film. The film was hydrated in a solution of 20 mM HEPES (pH 7.4). After heating overnight to 50 ° C followed by 10 cycles of freezing and thawing at a temperature of -177 ° C and 70 ° C, the colloidal solution was several times displaced through polycarbonate filters with pores with a diameter of 1 μm, 0.4 μm, 0.2 μm and 0.1 μm. Subsequently, a solution of GdCl ₃ (5 equivalents) in a 20 mM HEPES solution of pH 7.4 was added to the colloidal solution by polymers at 50 ° C for 2 hours. Subsequently, the polymersomes were dialyzed overnight to remove excess Gd (III). Dialysis was performed against a 20 mM HEPES solution of pH 7.4. The minimum average radius of polymers was determined using dynamic light scattering (DLS). The shape of the polymer vesicles was determined using cryo-TEM. Gadolinium concentration was determined using ICP-MS. The longitudinal and transverse relaxation periods (T ₁ and T ₂ ) were determined at 60 MHz.

Example 3

Aspherical polymersomes containing a compound with a chemical shift and paramagnetic complexes terminated using DOTA polymers (14).

A polymer vesicle with an average diameter of 100-150 nm was obtained using the method of hydrating a thin film, followed by displacement, as described in Example 1. In this case, the film was hydrated in a 20 mM HEPES solution (pH 7.4) containing 65 mM [Tm (hpdo3a) (H ₂ O)]. After heating to 50 ° C overnight, followed by ten freeze-thaw cycles at -177 ° C and 70 ° C, the colloidal solution was several times forced through a polycarbonate filter with a pore diameter of 1 μm, 0.4 μm, 0.2 μm and 0.1 microns. The obtained polymersomes (12) were dialyzed overnight to remove [Tm (hpdo3a) (H ₂ O)], which was not captured after hydration of the lipid film, and aspherical polymersomes were obtained (15). Dialysis was performed using 20 mM HEPES buffer containing 0.3 M NaCl. Then, a solution of TmCl ₃ (5 equivalents) in 20 mM HEPES buffer containing 0.3 M NaCl was added to the colloidal solution with polymers at 50 ° C for 2 hours. Polymersomes (14) were dialyzed overnight to remove excess Tm. Dialysis was performed against 20 mM HEPES buffer containing 0.3 M NaCl (pH 7.4). The minimum average radius by polymers (14) was determined using dynamic light scattering. The shape of the polymer vesicles was studied using cryo-TEM. Gadolinium concentration was determined using ICP-MS. The longitudinal and transverse relaxation periods (T ₁ and T ₂ ) were determined at 60 MHz.

Example 4

Radiolabeled Polymersomes and Emulsions

Obtaining a polymer-stabilized emulsion

The emulsion was prepared from octan-2-yl-2,3,5-triiodobenzonate (25% w / v) using 2% w / w poly (butadiene (l, 2 addition) -block poly (ethylene oxide) (f _EO 0 61; Mw _phil = 2033 g / mol; Mw _phob = 1305 g / mol) and 5 mol% DOTA-functionalized copolymer (5). The emulsion was prepared in 2.1 mM THAM buffer containing 152 mM NaCl at pH 7, 4 using a high pressure microfluidizer system (microfluidizer M110S, Microfluidics Int. Corp., Newton MA) at 70 ° C. Extensive dialysis was performed against THAM buffer (1 L) containing Chelex (2 g / L) for three days. Then stabilized with polymer em lsiyu filtered through a filter with pores of 450 nm.

Emulsion labeling

An emulsion stabilized with a DOTA copolymer (300 μl) was incubated with¹¹¹Incl₃ 30 MBq in 0.05 M HCl (4 μl) for 1 hour at 70 ° C. Then, free DTPA was added to the reaction mixture to remove free¹¹¹In. 1 μl of the reaction mixture was applied to a TLC plate coated with silica. As an eluent, a 200 mM EDTA solution containing 9.0 g / L NaCl was used. TLC was analyzed using a phosphoimager FLA-7000 (Fuji Film, Tokyo, Japan), and the introduction of radioactive particles was quantified using Aida software (Fuji film). The effectiveness of introducing a radioactive label using¹¹¹Incl₃30 MBq was 65%. The introduction of radioactive substances on a smaller scale (¹¹¹Incl₃ 4.6 MBq in 100 μl of emulsion) gave a yield of 97%. Although the output of this result is greater, 4.6 MBq is not enough for visualization. Thus, the described method with¹¹¹In 30 MBq was used for in-vivo studies. A radioactive isotope-labeled emulsion was tested on male Swiss mice (Charles River, Maastricht, Netherlands) for scanning using a dual-mode SPECT / CT. An isotope-labeled emulsion (200 μl) with an activity of 20.5 MBq was administered intravenously. Scanning using SPECT / CT was performed using NanoSPECT / CT (Bioscan).

The animal experiment was approved by the University Ethics Expert Committee for Animal Experiments at Maastricht University (Maastricht, Netherlands).

Claims (14)

1. Chelated amphiphilic polymer as a carrier capable of self-aggregation, containing a hydrophilic block and a hydrophobic block, where the hydrophilic block contains a chelating fragment as an end group,
where the hydrophilic block is selected from the group consisting of polyethylene oxide, polymethacrylic acid, polyacrylamide derivatives, polyhydric alcohols such as polyvinyl alcohol or polyhydroxyethyl methacrylate, hydrophilic polypeptides and sugar derivatives,
where the hydrophobic block is selected from the group comprising polybutadiene, polyisoprene, polyethylene and all polymers with a skeleton of carbon atoms and side groups of hydrophobic nature,
where the chelating moiety is selected from the group consisting of polyphosphates, aminocarboxylic acids, 1,3-diketones, hydroxycarboxylic acids, polyamines, amino alcohols, aromatic heterocyclic bases, phenols, aminophenols, oximes, peptides containing proximal chelate groups, Schiff bases, tetrap sulfur compounds, synthetic macrocyclic compounds, phosphonic acids, or combinations of two or more of the above compounds,
where the chelate amphiphilic polymer satisfies the general structural formula (i):

where X is a chelating moiety; M _A is a repeating hydrophilic unit forming a hydrophilic block; M _B is a repeating hydrophobic unit forming a hydrophobic block; n and m, each independently, are integers from 3 to 1,000,000, preferably from 5 to 5,000, representing the number of monomer units forming the corresponding blocks. 2. The chelated amphiphilic polymer according to claim 1, wherein the hydrophilic block is a poly (ethylene oxide) block, preferably with a weight average molecular weight of from 500 to 10,000. 3. The chelated amphiphilic polymer according to claim 1, where the hydrophobic block has a Tg below 70 ° C, and is preferably selected from the group consisting of poly (butadiene), poly (isoprene) and poly (ethylene). 4. The chelate amphiphilic polymer according to claim 1, wherein the chelating moiety is selected from the group consisting of DOTA, DTPA, HYNIC and desferoxamine. 5. A particle as a carrier containing a self-aggregating structure of a chelating amphiphilic polymer according to any one of the preceding claims. 7. The particle according to claim 6, containing a combination of chelate amphiphilic polymers according to any one of claims 1 to 4 and an amphiphilic non-chelating polymer. 7. The particle according to claim 6, where the non-chelating amphiphilic polymer has a poly (oxyethylene) chain as a hydrophilic block, the length of said chain is greater than the length of the hydrophilic block of the chelate amphiphilic polymer. 8. The use of particles according to any one of paragraphs.5-7 as a contrast agent for visualization. 9. A contrast agent for CEST MPT containing particles according to any one of claims 5 to 7, wherein the self-aggregating structure is polymersomes having a shell formed by a bilayer of one or more amphiphilic polymers, the bilayer contains a chelate amphiphilic polymer, where the shell is enclosed a cavity containing a stock of protons for analysis capable of diffusion through the shell, and where a chelating fragment of a chelate amphiphilic polymer extending toward the cavity is provided with chelating paramagnetic material. 10. The contrast agent for CEST MPT according to claim 9 with a non-spherical shape. 11. A contrast agent for SPECT or PET containing a particle according to any one of claims 5 to 7, wherein the self-aggregating structure is a polymer-stabilized oil-in-water emulsion, and where the chelating fragments of the chelate amphiphilic polymer are provided using a chelating radionuclide suitable for SPECT or PET. 12. A spectral CT contrast agent containing particles according to any one of claims 5 to 7, wherein the chelating moiety of the chelate amphiphilic polymer is provided using Gd. 13. A particle according to any one of claims 5 to 7, wherein one or more chelating moieties contains a metal ion in the form of a coordination complex, metal ions form chelate complexes inside. 14. The method of producing a particle according to item 13, where the chelate amphiphilic polymer must be in an aqueous medium in order to form a bilayer surrounding the cavity, and where the chelating fragments are subjected to the formation of coordination complexes with metal ions before the formation of the bilayer.

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